Screening methods using a novel human EP prostaglandin receptor

ABSTRACT

A gene encoding the HP4 human prostaglandin receptor is disclosed. The protein encoded by this gene exhibits significant sequence identity with other prostaglandin receptors. The HP4 receptor, when expressed in eukaryotic cells, is capable of binding prostaglandins and their analogs and stimulating adenylate cyclase activity in response to prostaglandins. Also disclosed are antisense agents able to decrease or prevent translation of a human HP4 prostaglandin receptor.

This application is a continuation-in-part of application Ser. No.09/019,393, filed Feb. 5, 1998, which was a divisional of applicationSer. No. 08/239,431, filed May 5, 1994, now issued as U.S. Pat. No.5,716,835.

FIELD OF THE INVENTION

This invention relates to the cloning and expression of a novel humanprostaglandin receptor. Methods of identifying compounds capable of bothbinding to and activating this receptor are also disclosed. Asdetermined using the disclosed methods, the receptor exhibits EP₂pharmacology.

BACKGROUND OF THE INVENTION

Prostaglandins are a group of hormone mediators derived from themetabolism of arachidonic acid via the cyclooxygenase enzymatic pathway.In the prostaglandin biosynthetic pathway, arachidonic acid is firstconverted to prostaglandin endoperoxide H2 (PGH₂) by PGH₂ synthasesfollowed by the cell-specific isomerization or reduction of PGH₂ to theactive prostaglandins: PGD₂, PGE₂, PGF_(2α), prostacyclin (PGI₂) andthromboxane (TXA₂). Following enzymatic conversion, the majorbiologically active prostaglandins exert their actions locally on thecells in which they were synthesized (autocrine) and/or on nearby cells(paracrine) through specific G protein-coupled receptors (Smith, (1992)Am. J. Physiol., 263: F181-F191) to either stimulate or inhibit theproduction of second messengers. Prostaglandins elicit a diversespectrum of often opposing biological effects including musclecontraction and relaxation, potentiation and inhibition of plateletaggregation, and vasodilation and vasoconstriction. Prostaglandins alsoexhibit both pro-inflammatory and anti-inflammatory effects. Theysynergize with other pro-inflammatory mediators such as leukotrienes andbradykinins, but attenuate interleukin-1 (IL-1) production and inhibitvarious aspects of leukocyte function (Giles, (1990) Trends Pharmacol.Sci., 11:301-304).

Prostaglandin E₂ (PGE₂) exhibits a broad range of actions in a number oftissues by binding to at least four EP receptor subtypes. It actsthrough pharmacologically distinct stimulatory (EP₂) and inhibitory(EP₃) receptor subtypes to stimulate and inhibit cyclic AMP (cAMP)formation, respectively (Sonnenburg, and Smith, (1988) J. Biol. Chem.,263: 6155-6160) . PGE₂ also stimulates calcium release and proteinkinase C activity in the rabbit kidney collecting tubule, most likely bybinding to the EP₁ receptor subtype which is coupled to stimulation ofphospholipase C (Hebert et al., (1990) Am. J. Physiol., 259: F318-F325).The EP₄ receptor is an additional subtype of PGE₂-sensitive receptorthat was recently identified based on agonist effects and blockade bythe antagonist AH 23848B (Louttit et al., (1992) The EighthInternational Congress on Prostaglandins and Related Compounds,Montreal, 258; Coleman et al., (1994) Prostaglandins, 47:151-168). OtherPGE₂-sensitive receptors with distinct agonist pharmacology have beendescribed (Milne et al., (1994) Br. J. Pharmacol., 111:79), but it isnot clear whether they are different from the EP₄ receptor.

Analogs of PGE₂ that are therapeutically useful will elicit or blockonly a subset of its actions by acting on a single EP receptor subtype.Because prostaglandin receptors are present in tissues in low abundance,the discovery of such analogs is facilitated by the cloning of thereceptors. Assigning cloned receptors to a correspondingpharmacologically defined binding site is an iterative process. Definingnovel subtypes requires selective compounds, which may only be developedonce the receptor is cloned.

Three human receptors that bind PGE₂ have been cloned. The EP₁ (Funk etal., (1993) J. Biol. Chem., 268: 26767-26772) and EP₃ (Regan et al.,(1994) Br. J. Phamacol.,112:377-385) subtypes have been wellcharacterized with subtype-selective compounds, but the pharmacology ofthe putative EP₂ receptor (An et al., (1993) Biochem. Biophys. Res.Commun., 197:263-270; Honda et al., (1993) J. Biol. Chem.,268:7759-7762) is not entirely consistent with the pharmacology derivedfrom tissue models of the EP₂ receptor. In particular, the EP₂-selectiveagonist butaprost, is inactive (Gardiner (1986) Br. J. Pharmacol.,87:45-56; Coleman, (1993) in Eicosanoids and Other Bioactive Lipids inCancer, Inflammation and Radiation Injury, Nigan et al., eds., pp.135-141). The pharmacology of this putative EP₂ clone is more similar tothat of the EP₄ receptor, but it was named before the EP₄ receptor hadbeen described.

The deduced protein sequences of the cloned receptors indicate that allare members of the G protein-linked receptor superfamily, having sevenputative membrane-spanning hydrophobic domains. The proteins sharesignificant amino acid sequence similarity with other members of thisfamily including the thromboxane (TP) receptor (Hirata et al., (1991)Nature 349: 617-620), rhodopsin and the adrenergic receptors.

The cloning of EP₂ and/or additional EP receptors will facilitateidentification of prostaglandins which can modulate specific effectselicited by this receptor. Since these effects will differ from thoseactivated by other EP receptors, such compounds will have therapeuticutility.

SUMMARY OF THE INVENTION

One embodiment of the present invention is an isolated DNA moleculeencoding a novel mammalian prostaglandin EP receptor, herein called HP4(Human Placental clone Number 4). Preferably, the DNA molecule is human;most preferably, the DNA molecule has the nucleotide sequence shown inSEQ ID NO: 3. According to another aspect of the invention, there isprovided an isolated DNA molecule having at least 18 consecutivenucleotides of the DNA molecule encoding the HP4 receptor. In accordancewith another aspect of the invention, there is provided an isolatedamino acid sequence derived from the HP4 receptor DNA sequence.Preferably, the amino acid sequence is human; most preferably it is SEQID NO:4. Advantageously, there is also provided a recombinant constructcomprising the HP4 receptor DNA sequence operably linked to aheterologous promoter. In another aspect of this preferred embodiment,there is provided an isolated antibody having binding affinity for theisolated HP4 receptor amino acid sequence. Preferably, the antibody ismonoclonal.

Another embodiment of the invention is a method of screening compoundsfor binding to the prostaglandin HP4 receptor comprising:

-   -   transfecting cells with a DNA molecule encoding an HP4 receptor,        wherein the DNA molecule is operably linked to a promoter in an        expression vector;    -   culturing the cells to express the HP4 receptor;    -   incubating at least the cell membranes of the cells in the        presence of a labeled compound to be tested for binding affinity        to the HP4 receptor; and    -   measuring the amount of label bound to the cell membranes,        wherein an increased amount of the label associated with the        cell membranes indicates that the compound binds to the        receptor.

Preferably, the cells are mammalian; most preferably, they are COS-7cells. In another aspect of this preferred embodiment, the HP4 receptoris human. Preferably, it is encoded by the polynucleotide of SEQ IDNO:3. Advantageously, the expression vector is mammalian; mostpreferably, it is pBC12BI. In accordance with this aspect of theinvention, the label is radioactive, calorimetric or fluorimetric.

In accordance with another aspect of the invention, there is provided amethod of determining the ability of a compound to inhibit ligandbinding to the prostaglandin HP4 receptor, comprising:

-   -   transfecting cells with a DNA molecule encoding a prostaglandin        HP4 receptor, wherein the DNA molecule is operably linked to a        promoter in an expression vector;    -   culturing the cells to express the HP4 receptor;    -   incubating at least the cell membranes of the cultured cells in        the presence of a labeled ligand having binding affinity for the        receptor and a test compound; and    -   determining the level of binding of the ligand to the        prostaglandin HP4 receptor in the presence of the compound,        wherein a lower level of ligand binding in the presence of the        compound indicates that the compound binds to the receptor.

Preferably, the cells are mammalian; most preferably, they are COS-7cells and the HP4 receptor is human. In another aspect of the invention,the HP4 receptor is encoded by the polynucleotide of SEQ ID NO:3.Advantageously, the compound label is radioactive, calorimetric orfluorimetric, the expression vector is mammalian, most preferablypBC12BI, and the ligand is PGE₂.

Still another embodiment of the invention is a method for identifyingcompounds that are agonists of the HP4 prostaglandin receptor,comprising:

-   -   transfecting cells with a DNA molecule encoding the HP4        receptor, wherein the DNA molecule is operably linked to a        promoter in an expression vector;    -   preincubating the cells in the presence of a phosphodiesterase        inhibitor;    -   incubating the cells in the presence or absence of a compound to        be tested;    -   lysing the cells; and    -   determining the amount of cyclic AMP produced, wherein an        increased amount of cyclic AMP indicates that the compound is an        agonist of the receptor.

Preferably, the cells are mammalian; most preferably, they are COS-7cells. In another aspect of this preferred embodiment, the HP4 receptoris human. In another particularly preferred embodiment, the HP4 receptoris encoded by the polynucleotide of SEQ ID NO:3 . Advantageously, theexpression vector is mammalian, most preferably pBC12BI and thephosphodiesterase inhibitor is isobutylmethylxanthine.

According to another aspect of this embodiment, there is provided a cellline in continuous culture expressing the HP4 prostaglandin receptor.Preferably, this HP4 prostaglandin receptor is human; most preferably itis encoded by SEQ ID NO:3. Advantageously, the cells are CHO cells.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B and 1C illustrate competition curves of ³H-PGE₂ binding toCOS-7 membranes from COS-7 cells transfected with the HP4 receptor cDNA.FIG. 1A compares the displacement of ³H-PGE₂ by the naturally-occurringprostaglandins PGE₂, PGE₁, PGD₂ and PGF₂α. FIG. 1B depicts competitionof ³H-PGE₂ binding by synthetic prostaglandins exhibiting selectivityfor the EP₂ receptor: A13205, Butaprost, 19 (R)-hydroxy PGE₂ and11-deoxy PGE₁. FIG. 1C compares the inhibition of radioligand binding byadditional PGE analogs 16,16-dimethyl PGE₂, MB 28767, sulprostone andPGE₁-1-OH. The y-axis indicates the percentage ³H-PGE₂ specificallybound and the x-axis indicates the concentration of competitor added(log M). Points represent the mean values±standard error from threeseparate experiments, performed in duplicate.

FIG. 2 shows the effects of prostaglandin treatment on cAMP levels inCOS-7 cells, after transient transfection with DNA encoding the humanHP4 receptor. Subsequent to transfection, cells were stimulated witheither PGE₂, AH13205, butaprost or PGE₁-1-OH. The drug concentration(−log M) is shown on the x-axis and the amount of cAMP produced(pmol/10⁵ cells) is shown on the y-axis.

DETAILED DESCRIPTION OF THE INVENTION

This invention discloses the cloning, sequencing and characterization ofa novel human EP prostaglandin receptor, referred to herein as HP4(Human Placental clone number 4). To characterize the pharmacology ofthe prostaglandin receptor of the present invention, the gene wastransfected into COS-7 cells which lack prostaglandin receptors andcompetition binding assays using tritiated PGE₂ were performed on theplasma membrane fraction (Sugimoto et al., (1992) J. Biol. Chem., 267:6463-6466). The results suggest that HP4 is a pharmacologicallycharacterized EP₂ receptor.

The HP4 gene sequence, fragments thereof, vectors containing thissequence or unique fragments thereof, cells transfected with thissequence or fragments thereof and protein purified from these cells willbe useful for studying the pharmacology and the cellular distributionand expression of the HP4 receptor. Since prostaglandins are known to beinvolved in a variety of biochemical processes including musclerelaxation, platelet aggregation, vasodilation, and inflammation, thereceptor of the present invention will be useful for determining thespecific processes mediated by the receptor. Since Northern blotanalysis demonstrated that HP4 was expressed at high levels in the lung(Example 5), the receptor of the present invention may be important inthe development of treatments for bronchopulmonary inflammation andasthma. Polymerase chain reaction (PCR) amplification of the HP4sequence indicated that it was present in leukocytes (Example 6),suggesting that it may play an important role in regulatinginflammation.

The present invention will also facilitate the identification ofcompounds which specifically bind to this newly-identified prostaglandinreceptor. Since this receptor will mediate responses different fromthose mediated by the other EP receptor subtypes, these compounds willhave utility as therapeutic agents. For example, the EP₂-selectiveagonist AH13205 has been shown to induce bronchodilation and inhibit therelease of the inflammatory mediator leukotriene B4 from humanneutrophils (Nials et al., (1993) Cardiovascular Drug Rev., 11:165-179).The compound also inhibits the spontaneous contraction of humanmyometrium (Yeardley et al., (1992) Br. J. Pharmacol. Proc. Suppl.,107:90P).

Fragments of the HP4 receptor gene consisting of at least 18 consecutivenucleotides unique to HP4 will be useful as probes and PCR primers forisolating other human prostaglandin receptors, for isolating thecorresponding receptor gene from other species and for determining HP4RNA expression in various human tissues. These oligonucleotides will beuseful for in situ hybridization and to probe Northern blots of RNAisolated from various tissues by well-known methods to determine the HP4receptor cellular distribution.

As specific subsets of the prostaglandin receptor family may be involvedin different cellular actions, it is important to identify the receptorsubtypes expressed by each cell. It can be appreciated that those ofordinary skill in the art could determine unique fragments of the humanHP4 receptor and use these fragments as probes to determine cellsexpressing the desired prostaglandin receptor gene.

In addition, DNA sequences of 18 nucleotides correspond to six aminoacids. Those of ordinary skill in the art will appreciate that a sixamino acid peptide, when coupled to an immunogenic carrier protein suchas keyhole limpet hemocyanin, can be utilized as an antigen to raiseantibodies against HP4 receptor epitopes. Alternatively, the HP4 cDNA orfragments thereof can be expressed and the resulting polypeptiderecovered and used as an immunogen. Antibodies against the HP4 receptorprotein will allow immunohisto-chemical localization of the protein incells, tissues and body fluids, thereby providing a means foridentification of cells expressing the HP4 receptor subtype.

The use of a number of eukaryotic expression vectors is within the scopeof the present invention. Those of ordinary skill in the art willappreciate that once the HP4 receptor clone has been identified andsequenced, it can rapidly be incorporated into almost any desiredvector. In the present invention, preferable expression vectors aremammalian, with the most preferable vector being pBC12BI. In addition,the use of yeast, baculovirus and prokaryotic expression vectors is alsowithin the scope of the present invention as is the production of HP4receptors or fragments thereof in these cell types.

Binding assays using the expressed protein, either in whole transfectedcells or in membrane preparations, will be particularly useful foridentifying HP4 receptor agonists and antagonists. Although thepreferred method of identifying receptor ligands is throughradiolabeling, other methods known in the art are also within the scopeof the present invention. For instance, well known methods exist forcolorimetrically and fluorimetrically labeling compounds. One can alsomeasure functional responses in cells expressing the HP4 receptorprotein by using signaling systems including, but not limited to,adenylate cyclase, phosphoinositide hydrolysis, guanylate cyclase, ionfluxes and pH changes. These types of response systems can either bepresent in the host cell or introduced into the host cell along with thereceptor. Although the transfected cells of the present invention aremammalian, any cell type able to express a transfected HP4 gene iscontemplated. Transient transfection of HP4 into cells is describedbelow; however, production of stable transfectants expressing the HP4gene using well-known methods is also contemplated (Example 9).

With the gene sequence determined, mutations can now be introduced tostudy structure-function relationships as they relate to ligand bindingand effector system coupling. For example, point mutations can beintroduced into the receptor at various locations by well-known methods.The mutant receptor can then be introduced into cells and the effect ofthe mutation on ligand binding and signaling pathways can be determined.This analysis will indicate which amino acid residue(s) are involved inligand binding and effector system coupling, helping to differentiatethe functions of EP receptor subtypes, and facilitating the discovery ofdrugs specific for the HP4 receptor. As a first step in isolating theHP4 receptor gene, a human placental cDNA library was screened under lowstringency hybridization conditions with the full-length coding sequenceof the human EP₃ receptor gene as described in the following example.This gene is described in U.S. patent application Ser. No. 08/155,005,filed Nov. 19, 1993, which is hereby incorporated by reference.

EXAMPLE 1 Cloning of the Human HP4 Receptor by Low StringencyHybridization

DNA encoding the complete coding sequence of the human EP₃ receptor waslabeled with ³²P-dATP using a nick-translation kit (Gibco-BRL,Gaithersburg, Md.) and used to screen a λgt11 human placenta cDNAlibrary (Clontech, Palo Alto, Calif.) by plaque hybridization analysis.The library was plated using E. coli Y109OR⁻ cells at a density ofapproximately 25,000 plaques per plate. A total of 16 plates (400,000plaques) were used from which impressions were taken using nylonmembranes (Colony Plaque Screen, DuPont/NEN). DNA was denatured in 0.5MNaOH, 1.5M NaCl, neutralized in 0.5M Tris-HCl, pH 8.0, 1.5M NaCl andbaked at 80° C. for 2 hours. Filters were prehybridized for 2 hours at37° C. in 50% deionized formamide, 1% sodium dodecyl sulfate (SDS), 1MNaCl, 100 μg/ml sonicated, boiled herring sperm DNA. The ³²P-labeledprobe (1×10⁶ cpm) was added and the filters were hybridized at 37° C.overnight. The filters were then washed for 1 hour at 45° C. in1×standard saline citrate (SSC), 0.1% SDS, air-dried and exposed toKodak XAR film (Eastman-Kodak, Rochester, N.Y.) overnight at −70° C.

Polymerase chain reaction (PCR) using primers complementary to λgt11sequences flanking the cDNA insert region (5′-GACTCCTGGAGCCG-3′; SEQ IDNO:1 and 5′-CGCGGCCAGCGATGG-3′; SEQ ID NO:2) and restriction analysiswere used to amplify seven related clones which were placed into threegroups based on their size. One member of each group was subcloned intothe EcoRI site of pBluescript (Stratagene, La Jolla, Calif.) and itsnucleotide sequence determined using the dideoxy chain terminationmethod (United States Biochemical, Cleveland, Ohio). The clonescontained overlapping nucleotide sequences. One clone, designated HP4,contained a 2296 nucleotide insert (SEQ ID NO:3) having 156 nucleotidesof 5′-untranslated sequence, an open reading frame of 1074 nucleotidesencoding a protein of 358 amino acids and 1066 nucleotides of3′-untranslated sequence. The below-referenced KS/HP4 plasmid has beendeposited with the American Type Culture Collection and assignedAccession No. 97472.

The HP4 deduced amino acid sequence was found to have seven hydrophobicputative membrane-spanning domains characteristic of G protein-coupledreceptors. The HP4 sequence was aligned with the various putativeintracellular loops, extracellular loops, transmembrane domains, andcarboxy terminal regions of the thromboxane (TP), EP₁, EP₂ and EP₃receptors. The alignment revealed a number of conserved residues presentin the aligned sequences. In particular, characteristic prostaglandinreceptor sequences in the second extracellular loop and seventhtransmembrane domain indicated that the isolated HP4 receptor was aprostaglandin receptor. The overall sequence identity of the putativetransmembrane regions of HP4 with those of other prostaglandin receptorsis 34% for human EP3A, 38% for murine EP2, 37% for murine EP1 and 31%for human TP. Thus, although HP4 possesses conserved sequence motifsfound in these previously identified receptors, it is clearly distinct.

A vector for the expression of HP4 in eukaryotic cells was made asfollows. PCR was used to amplify nucleotides 124-387 of KS/HP4 (thepBluescript HP4 clone). The primers used were5′-GATGAGCTCTTTAAAAGGAGGGCGCATCTCTTTTCCAGG-3′ (sense; SEQ ID NO:5) and5′-GGTGAACACCAGCTCGGT-3′ (antisense; SEQ ID NO:6). The PCR product wasdigested with SacI and ligated to the large fragment remaining from thedigestion of KS/HP4 with SacI. E. coli cells were transformed and aplasmid with a complete open reading frame in the sense orientation wasisolated. The latter was digested with DraI and ligated to the pBC12BIexpression vector which had been cleaved with BamHI and HindIII andfilled in with the large fragment of DNA polymerase I. E. coli cellswere transformed and a plasmid (pBC/HP4) was isolated in which the DraIsite adjacent to nucleotide 124 in HP4 was ligated to the HindIII site(nucleotide 314 in pBC12BI). This orientation placed the codingsequences of HP4 (nucleotides 157-1230) downstream (3′) of the roussarcoma virus promoter in pBC12BI. The final construct, therefore,contained 33 bases of HP4 5′-untranslated sequence, the coding region,and 15 bases of 3′-untranslated sequence.

So as to perform the necessary binding assays for demonstrating theligand specificity of the protein derived from the isolated clone, theHP4 receptor was expressed in transfected COS-7 cells as described inthe following example.

EXAMPLE 2 Expression of the Human HP4 Receptor in COS-7 Cell

Monolayers of COS-7 cells (ATCC CRL 1651; 70-80% confluent) were rinsedwith Phosphate Buffered Saline (PBS, Ca/Mg-free) in 150×25 mm culturedishes. Ten ml transfection mix, consisting of 5 μg/ml plasmid DNA and0.5 mg/ml DEAE-dextran in PBS, was added to each dish and cells wereincubated for 30 min at 37° C. Nine ml of the solution was removed fromeach dish followed by the addition of 10 ml of 100 mM chloroquine inDulbecco's Modified Eagle Medium (DMEM)/5% fetal bovine serum (FBS). Thecells were incubated for 2.5 hr at 37° C., the solution aspirated and 10ml of 10% dimethyl sulfoxide (DMSO) in DMEM/5% FBS was added. After a2.5 minute incubation at 37° C., the solution was aspirated and 30 mlDMEM/5% FBS was added. The cells were incubated at 37° C. with mediachanges at 24 and 48 hours. After 72 hours, the media was aspirated andthe cells were scraped into cold TME buffer (50 mM Tris-HCl, pH 7.4, 10mM MgCl₂, 1 mM EDTA). The dishes were rinsed with cold TME buffer andthe cells combined and placed on ice.

To demonstrate the binding of EP receptor ligands to isolated membranesof COS-7 cells expressing the HP4 receptor, membranes were isolated andthe binding of radiolabeled ligands was assessed in the presence ofincreasing concentrations of unlabeled prostaglandin receptor agonistsas described in the following example.

EXAMPLE 3 COS-7 Membrane Preparation and Radioligand Binding Assay

Transfected COS-7 cells were homogenized for 30 seconds at approximately80% power with a Brinkman PT 10/35 Polytron homogenizer. The resultinghomogenate was centrifuged at 19,000 rpm for 20 minutes at 4° C. using aSorvall SS-34 rotor. The membrane pellet was resuspended in cold TMEbuffer (1 ml per original dish), frozen in liquid nitrogen and stored at−80° C.

Membrane pellets were then diluted in ice-cold 50 mM Tris-HCl buffer atpH 7.4 using a sonicator set at 50 watts. Membrane suspensions (100 μl)were then added to each assay tube to start the binding reaction. Finalconcentrations of the competition assay were as follows: [³H]-PGE₂, 5nM; 100 μg protein/tube in a total volume of 200 μl Increasingconcentrations of compounds to be tested for competitively inhibitingbinding were incubated for 60 minutes at room temperature. Contents wereaspirated onto a presoaked ice-cold Whatman GF/B filter using a BrandelCell Harvester and washed three times with ice-cold assay buffer. Thefilters were dried, placed in scintillation fluid and counted.

As shown in FIG. 1A, the strongest competitors of [³H]-PGE₂ binding werePGE₂ itself and PGE₁, indicating that the HP4 receptor is in the EPreceptor class. Two highly selective agonists for the EP₂ receptor,AH13205 and butaprost (Coleman, 1993; Nials et al., 1993; Gardiner,1986), displaced [³H]-PGE₂ binding (FIG. 1B).

The inhibitory potency of AH13205 relative to PGE₂ is very similar totheir relative potencies in isolated smooth muscle preparations (Nialset al., 1993; Coleman et al., 1994) 19 (R)-hydroxy PGE₂ and 11-deoxyPGE₁, two additional prostanoids reported to exhibit some degree ofselectivity for the EP₂ receptor (Woodward et al., (1993)Prostaglandins, 46:371-383; Dong et al., (1986) Br. J. Pharmacol.,87:97-107), also exhibited competitive activity (FIG. 1B). FIG. 1C showsthat the EP₃-selective agonist MB 28767, was only slightly active, aswas PGE₁-1-OH. Sulprostone, an agonist at both the EP₁ and EP₃ receptors(Coleman, (1993)), was inactive. 16,16-dimethyl PGE₂, which stimulatesthe EP₄ receptor in the rabbit jugular vein (Milne et al., 1994), wasactive. These results are consistent with the HP4 receptor being thepharmacologically defined EP₂ receptor rather than the cloned receptorsthat were previously designated EP₂ (An et al., 1993; Honda et al.,1993). However, identity of HP4 with the EP₄ receptor or an additionalEP receptor cannot be ruled out.

The native EP₂ receptor is coupled through a G protein to thestimulation of adenylate cyclase, an enzyme which transiently associateswith the G protein upon prostaglandin binding and converts ATP to cAMP.To determine whether the cloned HP4 receptor could bind prostaglandinsand mediate changes in cAMP levels, the HP4 cDNA was transfected intoCOS-7 cells as described in Example 2. The effect of PGE2 andprostaglandin analogs on cAMP accumulation is described in the followingexample.

EXAMPLE 4 PGE₂ Treatment and cAMP Assay on HP4-Transfected COS Cells

HP4-transfected COS-7 cells, prepared as described in Example 2, werecultured in DMEM containing 10% FCS, 100 units/ml penicillin, 100 μg/mlstreptomycin. Cells were plated in 24 well plates (Falcon Labware,approximately 2 cm²/well) 24 hours after transfection. Cells weretrypsinized and resuspended in a small volume of medium (2-3 ml),counted using a hemocytometer and diluted to 7-8×10⁴ cells/ml (DMEM/5%FCS). One ml cell suspension was added to each well. After 24 hours at37° C., the medium was changed and on the following day, cells wererinsed briefly with 1 ml of serum-free medium and were pre-incubated for1 minute with 400 μl/well of serum-free medium containing 100 μg/mlisobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase, anenzyme which degrades cAMP. One hundred μl of the indicatedconcentrations (FIG. 2) of PGE₂ or prostaglandin analogs were added toeach well for 3 minutes at 37° C. Drug solutions were aspirated and 150μl TE solution (50 mM Tris-HCl, pH 7.5, 4 mM EDTA) was added. Cells werescraped into TE, boiled for 5 min. in microcentrifuge tubes and placedon ice. Samples were then centrifuged for 10 min at 14,000 rpm to obtaina clarified cytosolic fraction. The amount of cAMP was quantified asfollows. Assay standards of 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32 and64 pmol cAMP were prepared by diluting 2×concentrations 1:1 with TE.Fifty μl assay standard or cytosol was then combined with 50 μl[³H]-cAMP. One hundred μl (6 μg) protein kinase A (PKA), an enzyme whichbinds cAMP, was then added to each tube. One control tube received noPKA, only 100 μl 0.1% BSA/Tris. Samples were vortexed, placed on ice fortwo hours and 100 μl 2% BSA-2.6% charcoal suspension was added whichwill bind the PKA-cAMP complex, but not the free cAMP. Samples werevortexed, centrifuged for 45 seconds, placed on ice and 200 μlsupernatant transferred to scintillation vials for counting. Counts inthe drug-treated samples were compared to counts in the standard tubesto determine the amounts of cAMP. More [³H]-cAMP remaining in thesupernatant indicates that more cAMP was synthesized in response to thedrug and competitively inhibits binding of the labeled cAMP to PKA.

The results indicated that PGE₂ treatment resulted in significantlyincreased cAMP levels in HP4-transfected COS cells (FIG. 2). PGE₂ couldpotently stimulate cAMP formation followed by AH13205, butaprost andPGE₁-1-OH, in decreasing order of potency. These results are similar tothe results obtained in the radioligand binding studies and lend furthersupport to the pharmacological similarity of HP4 with thepharmacologically defined EP₂ subtype.

The expression of HP4 mRNA in various human tissues was then determinedas described below.

EXAMPLE 5 Tissue Distribution of the HP4 Prostaglandin Receptor Gene

A multiple human tissue Northern blot (Clontech, Palo Alto, Calif.)consisting of RNA isolated from heart, brain, placenta, lung, liver,kidney and pancreas was prehybridized in 4.4.times.SSPE, 44% deionizedformamide, 8.8× Denhardt's solution, 1.75% SDS and 88 μg/ml denaturedherring sperm DNA at 42° C. with constant rotation. The prehybridizationsolution was removed and the filter was incubated in fresh solutioncontaining 1.5×10⁶ cpm/ml nick-translated HP4 cDNA prepared with a kit(Gibco BRL, Gaithersburg, Md.) at 42° C. overnight with constantrotation. The blot was washed six times in 2×SSC, 0.5% SDS at roomtemperature for 5 minutes each with constant agitation. The blot wasthen washed in 0.1×SSC, 0.1% SDS at 50° C. for 40 minutes with onechange of solution, dried and exposed to x-ray film. The lanescontaining the placenta and lung RNA were strongly positive. Since thisreceptor is expressed at high levels in the lung, it may play a role inregulating airway opening and inflammation in respiratory disorders suchas asthma and emphysema.

Because the human uterus (Senior et al., (1991) Br. J. Pharmacol.,102:747-753) and human neutrophils (Nials et al., 1993) are EP₂-activetissues, as well as the lung, PCR amplification of RNA was thenperformed to determine whether HP4 was expressed in human uterus, humanplacenta, human promyelocytic leukemia HL60 cells (ATCC CCL 240) and/orhuman acute T-cell leukemia Jurkat T-cells (ATCC TIB 152) as describedbelow.

EXAMPLE 6 PCR Amplification of HP4 From Human Cells and Tissues

Sense (5′-CTTACCTGCAGCTGTACG-3′; SEQ ID NO:7) and antisense(5′-GATGGCAAAGACCCAAGG-3′; SEQ ID NO:8) primers corresponding to thesecond extracellular loop and the seventh transmembrane region,respectively, of the human HP4 receptor clone were used in PCRreactions. Control sense (5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′; SEQ IDNO:9) and antisense (5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC; SEQ ID NO:10)primers to β-actin were also used. RNA was isolated from tissues andcell lines by techniques well known in the art.

The PCR reactions contained 1 μl of the RT reaction, 36.5 μl water, 5 μl10× buffer (Perkin-Elmer, Norwalk, Conn.), 3 μl MgCl2 (25 mM), 2 μldNTPs (1.25 mM each), 1 μl tetramethyl ammonium chloride (TMA; 2.5 mM),0.625 μl sense primer (25 μM), 0.625 μl antisense primer (25 μM), 0.25μl Taq polymerase (Perkin-Elmer, 5 units/μl) in a final volume of 50 μl.The denaturation step was performed at 94° C. for 2 min, followed by35-40 cycles at 94° C. for 15 sec, 60° C. for 15 sec and 72° C. for 2min. A 6 min incubation at 72° C. completed the reaction.

Half of the PCR reactions (25 μl) were analyzed by electrophoresis on a1.5% agarose gel and staining with ethidium bromide. These samples wererun in parallel with positive controls using plasmid DNA as a templateand negative controls which did not contain a DNA template. A fragmenthaving the predicted molecular weight based on the HP4 sequence (368base pairs) was observed in RNA isolated from placenta, uterus, HL 60cells and Jurkat T-cells. No fragments were amplified in the absence ofa DNA template. The HP primers do not amplify a product from humangenomic DNA, probably because there is an intron in between the twoprimers. Thus, the PCR products reflect the presence of MRNA encodingHP4 and not of contaminating genomic DNA.

To estimate the relative level of HP expression from these sources, thesame cDNAs were amplified for 25-30 cycles using the actin primers, SEQID NOS:9 and 10. Comparison of the product yields with actin and HPprimers shows that HP4 is expressed in human uterus, placenta and HL-60cells, but only at low levels in Jurkat T-cells. Thus, this receptor mayalso regulate inflammatory processes in disorders such as emphysema andarthritis.

To further characterize the human HP4 receptor, polyclonal antibodiesare generated as described in the following example.

EXAMPLE 7 Production of Polyclonal Antibodies Against the Human HP4Receptor

PCR primers are used to amplify an approximately 126 nucleotide regioncorresponding to the hydrophilic amino acid segments connecting thefifth and sixth membrane spanning domains of the human HP4 receptor. Theresulting PCR product is purified by agarose gel electrophoresis, clonedinto an expression plasmid such as pGEX (Pharmacia, Piscataway, N.J.)and used to transform E. coli by standard procedures. The positiveclones are identified and induced to express the fusion protein, whichis purified by well known methods.

The purified fusion protein is injected into the breast muscle ofchickens (50-100 μg/injection) with booster injections given at two weekintervals. The IgY antibodies are purified from the egg yolks by wellknown methods and their specificity determined by immunoblotting oftissue extracts.

In addition, monoclonal antibodies to the human HP4 receptor can beprepared as discussed below.

EXAMPLE 8 Production of Monoclonal Antibodies Against the Human HP4Receptor

The HP4 receptor-transfected COS-7 cell lysate, isolated as described inExample 7, is centrifuged to isolate membranes. The isolated membranesare injected in Freund's complete adjuvant into mice. After 9 boosterinjections over a three week period, the spleens are removed andresuspended in PBS. The resuspended spleen cells are mixed(approximately 4:1) with SP2/0 myeloma cells. Polyethylene glycol isadded to fuse the myeloma and spleen cells, and the hybridomas areselected in HAT medium. The fused cells are aliquoted to allow growth ofonly one cell in each well of a 96 well microtiter plate. Each cell isexpanded, the media removed and secreted proteins are labeled with ¹²⁵I. The labeled media from each well is used to probe a Western blot oftransfected and untransfected COS-7 cell membranes.

The desired hybridoma produces a monoclonal antibody that strongly bindsa protein band in a transfected COS-7 cell membrane lane on a Westernblot, but does not bind to any other protein in that lane or in anuntransfected COS-7 cell membrane lane (control). This method can beused to detect those cells expressing the human HP4 receptor.

EXAMPLE 9

Production of Stably-Transfected Cells

To produce CHO cells stably transfected with the human HP4 gene, CHOcells are cotransfected with 10-30 μg human HP4 and 1-5 μg pSV2Neocarrying the neomycin resistance gene by calcium phosphate precipitation(Graham and Van der Eb, (1973) Virology, 52: 456-467). The cells arethen subjected to selection with 600 μg/ml genetecin (G418; Gibco). Theresistant colonies are selected, expanded and screened for receptorexpression using [³H]-PGE₂ binding as described in Example 3.

A murine homolog of the human HP4 prostaglandin receptor gene isisolated as described below.

EXAMPLE 10 Isolation of a Murine HP4 Prostaglandin Receptor Gene

The HP4 gene, isolated as described in Example 1, is digested withrestriction enzymes by well-known methods to obtain a DNA segment ofapproximately 1-1.5 kilobases. This segment is nick-translated using akit (Gibco BRL, Gaithersburg, Md.) and [³²P]-dATP, then used to screenmouse cDNA libraries which are available from several commercial sourcesincluding Clontech (Palo Alto, Calif.). The positive clones aresequenced and aligned with the HP4 sequence using one of a number ofcomputer sequence alignment programs well-known in the art to determinewhether the mouse clone shares significant sequence identity with humanHP4.

EXAMPLE 11 Antisense Oligonucleotides Directed to Human HP4Prostaglandin Receptor

Antisense agents directed to human HP4 prostaglandin receptor mRNA mayused to attenuate the effects of endogenous HP4 receptor agonists inpatients having conditions including, without limitation, chronic asthmaor immunosuppression. Such antisense agents include oligonucleotidesthat comprise “native” deoxyribonucleotides, or that may comprisemodified nucleotides. Modified nucleotides are monomeric compounds notusually (or ever) found in nature which have the ability to formhydrogen-bonded base pairs with a nucleotide base and are further ableto be heteropolymerized into a linear oligonucleotide or oligonucleotideanalog. Examples of modified bases include, for example,2′methoxyribonucleotides, methylphosphonate nucleotides, andphosphorothioate nucleotides. Additionally, antisense agents maycomprise oligonucleotide analogs such as peptide nucleic acids (PNAs);for the purpose of this application, PNAs and similar oligonucleotideanalogs shall be considered to consist of modified nucleotides. Numerousadditional modified nucleotides exist and are known to the person ofskill in the art; such additional modified nucleotides are intended tofall within the scope of this term. As used herein, the term“oligonucleotide” shall include oligonucleotide analogs such as thosementioned above, and shall mean a short linear molecule having theability to form hydrogen bonds with the nucleotide bases of a specificsegment of a single stranded nucleic acid molecule. Depending upon thespecific type of modification, modified antisense agents may be moreresistant to nuclease digestion, may have a greater melting temperature(T_(m)), and/or a greater mRNA specificity than oligonucleotides made ofunmodified nucleotides. These properties can result in an increasedability to prevent expression of a target protein. The antisense agentsdescribed herein function to bind HP4 receptor DNA and/or mRNA. and toprevent transcription or translation by any of a number of mechanismssuch as the recruitment of RNase H activity to a DNA:RNA hybrid withsubsequent destruction of the RNA or through steric hindrance by theantisense agent of ribosome access to the 5′ translation initiationregion of the target gene.

Antisense oligonucleotides may be delivered to the target cell as the“naked” oligonucleotide by injection, infusion, or inhalation into thetarget tissue. However (particularly in the case of charged antisenseagents) entry of the antisense agents into the cell through the apolarportions of the cell membrane can be problematic, and can result in adecrease in delivery and effectiveness. To overcome this problem,alternative delivery strategies have been devised.

One such strategy involves encasing the antisense agent in a liposome.Liposomes are artificial membrane analogs composed of lipids havingpolar headgroups. Particularly preferred for negatively chargedantisense agents are liposomes containing cationic lipids, whichfunction to neutralize the charge of the antisense agent and areattracted by the negatively charged outer plasma membrane. Additionally,liposomes may contain other types of lipids such as sterols (e.g.vitamin D₃ or cholesterol). The liposomes are formed by sonication of alipid-containing solution or extrusion of the solution through amicrobial filter. The advantage of using liposomes for drug delivery isthat the liposome can fuse with the plasma membrane or be taken up bythe cell through endocytosis, resulting in the liberation of theantisense agent within the cytoplasm of the cell. Certain pH-sensitivelipids, such as DOPE (dioleoylphosphatidyl-ethanolamine) willdestabilize at acidic pH, thus aiding in the liberation of the antisenseagent within the cell. The person of ordinary skill in the art will befamiliar with liposome technology; additionally a large number ofreferences are available in the scientific literature that deal withstrategies for drug delivery using liposomes. See e.g., Felgner et al.,U.S. Pat. No. 5,264,618, which is incorporated herein by reference. Suchtherapeutic agents in liposome formulations may be delivered by meansincluding injection, infusion, inhalation, and topical application.

An alternative strategy for the delivery of antisense agents is the useof vectors, such as vectors derived from a virus, which will deliver atranslatable nucleic acid region containing a selected short portion ofthe target nucleic acid sequence to the cell along with a strongpromoter, and permit the transcription of this nucleic acid region toform multiple complementary single-stranded antisense RNA copiesthereof. These RNA molecules would then act to hybridize with HP4 mRNA,thus preventing translation of the mRNA.

Depending upon the nature of the vector and its size, it could also beengineered to contain a foreign, specific RNA polymerase gene, such as,without limitation, the T4 bacteriophage RNA polymerase gene. Since T4RNA polymerase is quite fastidious in its specificity for its ownpromoter, in such a system the transcriptional effects of the vectorcould be effectively limited to the desired therapeutic effect byplacing the translatable nucleic acid region under control of a 5′ T4promoter sequence. The inclusion of the T4 RNA polymerase gene withinthe vector, and expression of T4 RNA polymerase within the host cell,would ensure that a large number of copies of the antisense agents wouldbe produced from each vector molecule. Other RNA polymerase genes havingstrong specificity for particular promoter sequences may also be used,and are known to those of skill in the art.

Viral vectors may be derived from viruses such as, without limitation,adenovirus, adeno-associated virus (AAV-2), and various retroviruses. Ofcourse, other applicable viral vectors are available or can beenvisioned by the person of ordinary skill in the art; the vectorsmentioned herein are by way of illustration rather than limitation.

Each prospective vector has its own benefits and deficits. For example,adenovirus infections are common and relatively benign in humans; thisvirus is one of those responsible for the common cold. The viruscontains a double-stranded DNA genome. After deletion of non-essentialgenes, the virus is able to carry about 8 kilobase pairs of an exogenousdouble-stranded DNA insert. This amount would be more than adequate tocarry the antisense agent or its complementary template as well asnecessary regulatory sequences, such as a strong promoter. However, theimmunogenicity of adenovirus is relatively high. Additionally,adenovirus does not stably integrate into the host chromosome, andtherefore its therapeutic effect is relatively transient; of course,this result may be advantageous when it is desired that the therapeuticeffect of the antisense agent be temporary. Certain constructs ofadenovirus (and other gene transfer vectors) have been made “replicationdeficient” in order to control the extent. and duration of infection,and to minimize the spread of the recombinant virus.

AAV-2 also commonly infects humans but is not known to cause a disease.The virus is quite small, and therefore it is relativelynon-immunogenic. However, the small size also means that there is lessroom for packaging a therapeutic nucleic acid sequence region and anynecessary regulatory sequences or genes such as an RNA polymerase gene.Wild-type AAV-2 stably integrates at a specific site in human chromosome19, however the gene responsible for this stable integration is deletedin recombinant versions of the viral genome, and this property istherefore lost. Over a period of time recombinant AAV-2 appears torandomly integrate into the host chromosome. Also, optimal geneexpression is only seen after 3-5 weeks when using such vectors.

Retroviruses such as modified Moloney murine leukemia virus have alsobeen used as the raw materials of engineered transfer vectors. The virusgives rise to a minimal immunological response. Retroviral vectorsspecifically infect dividing cells, and for this reason they appear asattractive candidates as vectors against cancers. Moreover, retroviralvectors stably integrate into the chromosomes of the host cell,providing the potential for long term expression of the passengernucleic acid, and thus reducing the need for frequent re-introduction ofthe vector. Construction of retroviral vectors has involved removal ofthe gag, pol, and env genes from the DNA provirus to make room for thegene(s) of therapeutic interest, up to about 8 kilobases of insertednucleic acid. This process makes the vector replication-deficient, andthe virus particles are propagated in special “packaging” cell linesthat contain the genes missing from the vector. See e.g., ThePharmacological Basis of Therapeutics Ch. 5 (Hardman et al. ed., 9^(th)ed. 1996), the disclosure of which is hereby incorporated by referenceas part of this disclosure.

Thus, in one aspect of the present invention, a patient having severechronic asthma is treated with a liposome composition containing an HP4prostaglandin receptor-directed antisense agent in an inhalant asfollows. A preparation of a synthetic oligonucleotide analog containing2′ O methylribofuranosyl (“2′methoxy”) residues is prepared. Theantisense agent is constructed to be able to hybridize with the 10nucleotides immediately 5′ to the first ATG codon of the coding sequencefor the HP4 receptor, and with the first 12 nucleotides of the codingregion of the HP4 gene. General methods for synthesizingoligonucleotides are well known, and methods for synthesizingoligonucleotide analogs containing 2′methoxyribonucleotides aredisclosed in, e.g., Becker et al., WO 98/02582, which is herebyincorporated by reference herein in its entirety.

A liposome preparation is made as follows: a total of 80 micromoles(total lipids) of a mixture of lipids including DOPE(dioleoylphosphatidyl-ethanolamine), oleic acid and Vitamin D₃ is madein an organic solvent in a molar ratio of 10:5:2, then the preparationis lyophilized. The lyophilizate is mixed with an aqueous solution ofthe purified oligonucleotide. Liposomes are formed by extrusion of themixture through a 5 micron filter.

The liposome solution is then delivered once daily for one week using anatomizing inhaler. The combination of inhaler delivery volume andconcentration of the liposomal solution is designed to deliver aneffective dose of the therapeutic antisense agent to lung tissue, thusresulting in the modulation of eosinophil recruitment to lung tissue andof contraction of the smooth muscle of the airways. Upon examination ofthe patient one week following the initiation of treatment, airwayinflammation and bronchiospasm is decreased as compared to a patientreceiving only a placebo control.

Other applications and modes of delivery of antisense agents will beapparent to those of skill in the art.

While particular embodiments of the invention have been described indetail, it will be apparent to those skilled in the art that theseembodiments are exemplary, rather than limiting, and the true scope ofthe invention is that defined in the following claims.

1-10. (canceled)
 11. A method of screening compounds able to bind to theprostaglandin HP4 receptor, comprising the steps: a) culturing cellsexpressing the HP4 receptor, b) incubating the expressed HP4 receptor inthe presence of a compound suspected of being able to bind saidreceptor, c) directly or indirectly detecting the formation of a complexcomprising said HP4 receptor and said compound as an indication of theability of said compound to bind said receptor.
 12. The method of claim11 wherein said HP4 receptor comprises an amino acid sequence encoded byplasmid KS/HP4.
 13. The method of claim 12 wherein said HP4 receptorconsists of said amino acid sequence.
 14. The method of claim 11 furthercomprising the step of incubating cell membranes of said cultured cellsin the presence of a ligand having binding affinity for said receptor,and detecting or measuring displacement of said ligand from saidisolated membranes in the presence of said compound as an indication ofthe ability of the compound to bind the HP4 receptor.
 15. The method ofclaim 11 further comprising a method for detecting an agonist of saidHP4 receptor, and further comprising the steps: incubating said cells inthe presence of said compound, and comparing production of cyclic AMP insaid cells with cyclic AMP production in control cells not incubatedwith said compound, wherein increased production of cyclic AMP in saidcells incubated with said compound indicates the ability of saidcompound to bind and stimulate the activity of said HP4 receptor. 16.The method of claim 14 wherein said HP4 receptor comprises an amino acidsequence encoded by plasmid KS/HP4.
 17. The method of claim 15 whereinsaid HP4 receptor comprises an amino acid sequence encoded by plasmidKS/HP4. 18-20. (canceled)